Erdenebelig Uyangaa

Distinct dictation of Japanese encephalitis virus-induced neuroinflammation and lethality via triggering TLR3 and TLR4 signal pathways.

Japanese encephalitis (JE) is major emerging neurologic disease caused by JE virus. To date, the impact of TLR molecules on JE progression has not been addressed. Here, we determined whether each TLR modulates JE, using several TLR-deficient mouse strains (TLR2, TLR3, TLR4, TLR7, TLR9). Surprisingly, among the tested TLR-deficient mice there were contrasting results in TLR3−/− and TLR4−/− mice, i.e. TLR3−/− mice were highly susceptible to JE, whereas TLR4−/− mice showed enhanced resistance to JE. TLR3 ablation induced severe CNS inflammation characterized by early infiltration of inflammatory CD11b+Ly-6Chigh monocytes along with profoundly increased viral burden, proinflammatory cytokine/chemokine expression as well as BBB permeability. In contrast, TLR4−/− mice showed mild CNS inflammation manifested by reduced viral burden, leukocyte infiltration and proinflammatory cytokine expression. Interestingly, TLR4 ablation provided potent in vivo systemic type I IFN innate response, as well as ex vivo type I IFN production associated with strong induction of antiviral PRRs (RIG-I, MDA5), transcription factors (IRF-3, IRF-7), and IFN-dependent (PKR, Oas1, Mx) and independent ISGs (ISG49, ISG54, ISG56) by alternative activation of IRF3 and NF-κB in myeloid-derived DCs and macrophages, as compared to TLR3−/− myeloid-derived cells which were more permissive to viral replication through impaired type I IFN innate response. TLR4 ablation also appeared to mount an enhanced type I IFN innate and humoral, CD4+ and CD8+ T cell responses, which were mediated by altered immune cell populations (increased number of plasmacytoid DCs and NK cells, reduced CD11b+Ly-6Chigh monocytes) and CD4+Foxp3+ Treg number in lymphoid tissue. Thus, potent type I IFN innate and adaptive immune responses in the absence of TLR4 were closely coupled with reduced JE lethality. Collectively, these results suggest that a balanced triggering of TLR signal array by viral components during JE progression could be responsible for determining disease outcome through regulating negative and positive factors.

CCR5 ameliorates Japanese encephalitis via dictating the equilibrium of regulatory CD4 + Foxp3 + T and IL-17 + CD4 + Th17 cells

BackgroundCCR5 is a CC chemokine receptor involved in the migration of effector leukocytes including macrophages, NK, and T cells into inflamed tissues. Also, the role of CCR5 in CD4+Foxp3+ regulatory T cell (Treg) homing has recently begun to grab attention. Japanese encephalitis (JE) is defined as severe neuroinflammation of the central nervous system (CNS) following infection with mosquito-borne flavivirus JE virus. However, the potential contribution of CCR5 to JE progression via mediating CD4+Foxp3+ Treg homing has not been investigated.MethodsInfected wild-type (Ccr5+/+) and CCR5-deficient (Ccr5−/−) mice were examined daily for mortality and clinical signs, and neuroinflammation in the CNS was evaluated by infiltration of inflammatory leukocytes and cytokine expression. In addition, viral burden, NK- and JEV-specific T cell responses were analyzed. Adoptive transfer of CCR5+CD4+Foxp3+ Tregs was used to evaluate the role of Tregs in JE progression.ResultsCCR5 ablation exacerbated JE without altering viral burden in the extraneural and CNS tissues, as manifested by increased CNS infiltration of Ly-6Chi monocytes and Ly-6Ghi granulocytes. Compared to Ccr5+/+ mice, Ccr5−/− mice unexpectedly showed increased responses of IFN-γ+NK and CD8+ T cells in the spleen, but not CD4+ T cells. More interestingly, CCR5-ablation resulted in a skewed response to IL-17+CD4+ Th17 cells and correspondingly reduced CD4+Foxp3+ Tregs in the spleen and brain, which was closely associated with exacerbated JE. Our results also revealed that adoptive transfer of sorted CCR5+CD4+Foxp3+ Tregs into Ccr5−/− mice could ameliorate JE progression without apparently altering the viral burden and CNS infiltration of IL-17+CD4+ Th17 cells, myeloid-derived Ly-6Chi monocytes and Ly-6Ghi granulocytes. Instead, adoptive transfer of CCR5+CD4+Foxp3+ Tregs into Ccr5−/− mice resulted in increased expression of anti-inflammatory cytokines (IL-10 and TGF-β) in the spleen and brain, and transferred CCR5+ Tregs were found to produce IL-10.ConclusionsCCR5 regulates JE progression via governing timely and appropriate CNS infiltration of CD4+Foxp3+ Tregs, thereby facilitating host survival. Therefore, this critical and extended role of CCR5 in JE raises possible safety concerns regarding the use of CCR5 antagonists in human immunodeficiency virus (HIV)-infected individuals who inhabit regions in which both HIV and flaviviruses, such as JEV and West Nile virus, are endemic.

CD11c hi Dendritic Cells Regulate Ly-6C hi Monocyte Differentiation to Preserve Immune-privileged CNS in Lethal Neuroinflammation

Although the roles of dendritic cells (DCs) in adaptive defense have been defined well, the contribution of DCs to T cell-independent innate defense and subsequent neuroimmunopathology in immune-privileged CNS upon infection with neurotropic viruses has not been completely defined. Notably, DC roles in regulating innate CD11b+Ly-6Chi monocyte functions during neuroinflammation have not yet been addressed. Using selective ablation of CD11chiPDCA-1int/lo DCs without alteration in CD11cintPDCA-1hi plasmacytoid DC number, we found that CD11chi DCs are essential to control neuroinflammation caused by infection with neurotropic Japanese encephalitis virus, through early and increased infiltration of CD11b+Ly-6Chi monocytes and higher expression of CC chemokines. More interestingly, selective CD11chi DC ablation provided altered differentiation and function of infiltrated CD11b+Ly-6Chi monocytes in the CNS through Flt3-L and GM-CSF, which was closely associated with severely enhanced neuroinflammation. Furthermore, CD11b+Ly-6Chi monocytes generated in CD11chi DC-ablated environment had a deleterious rather than protective role during neuroinflammation, and were more quickly recruited into inflamed CNS, depending on CCR2, thereby exacerbating neuroinflammation via enhanced supply of virus from the periphery. Therefore, our data demonstrate that CD11chi DCs provide a critical and unexpected role to preserve the immune-privileged CNS in lethal neuroinflammation via regulating the differentiation, function, and trafficking of CD11b+Ly-6Chi monocytes.

Amelioration of Japanese encephalitis by blockage of 4-1BB signaling is coupled to divergent enhancement of type I/II IFN responses and Ly-6C hi monocyte differentiation

BackgroundJapanese encephalitis (JE), a neuroinflammation caused by zoonotic JE virus, is the major cause of viral encephalitis worldwide and poses an increasing threat to global health and welfare. To date, however, there has been no report describing the regulation of JE progression using immunomodulatory tools for developing therapeutic strategies. We tested whether blocking the 4-1BB signaling pathway would regulate JE progression using murine JE model.MethodsInfected wild-type and 4-1BB-knockout (KO) mice were examined daily for mortality and clinical signs, and neuroinflammation in the CNS was evaluated by infiltration of inflammatory leukocytes and cytokine expression. In addition, viral burden, JEV-specific T cell, and type I/II IFN (IFN-I/II) innate responses were analyzed.ResultsBlocking the 4-1BB signaling pathway significantly increased resistance to JE and reduced viral burden in extraneural tissues and the CNS, rather than causing a detrimental effect. In addition, treatment with 4-1BB agonistic antibody exacerbated JE. Furthermore, JE amelioration and reduction of viral burden by blocking the 4-1BB signaling pathway were associated with an increased frequency of IFN-II-producing NK and CD4+ Th1 cells as well as increased infiltration of mature Ly-6Chi monocytes in the inflamed CNS. More interestingly, DCs and macrophages derived from 4-1BB KO mice showed potent and rapid IFN-I innate immune responses upon JEV infection, which was coupled to strong induction of PRRs (RIG-I, MDA5), transcription factors (IRF7), and antiviral ISG genes (ISG49, ISG54, ISG56). Further, the ablation of 4-1BB signaling enhanced IFN-I innate responses in neuron cells, which likely regulated viral spread in the CNS. Finally, we confirmed that blocking the 4-1BB signaling pathway in myeloid cells derived from hematopoietic stem cells (HSCs) played a dominant role in ameliorating JE. In support of this finding, HSC-derived leukocytes played a dominant role in generating the IFN-I innate responses in the host.ConclusionsBlocking the 4-1BB signaling pathway ameliorates JE via divergent enhancement of IFN-II-producing NK and CD4+ Th1 cells and mature Ly-6Chi monocyte infiltration, as well as an IFN-I innate response of myeloid-derived cells. Therefore, regulation of the 4-1BB signaling pathway with antibodies or inhibitors could be a valuable therapeutic strategy for the treatment of JE.

Ablation of CD11c(hi) dendritic cells exacerbates Japanese encephalitis by regulating blood-brain barrier permeability and altering tight junction/adhesion molecules.

Japanese encephalitis (JE), characterized by extensive neuroinflammation following infection with neurotropic JE virus (JEV), is becoming a leading cause of viral encephalitis due to rapid changes in climate and demography. The blood-brain barrier (BBB) plays an important role in restricting neuroinvasion of peripheral leukocytes and virus, thereby regulating the progression of viral encephalitis. In this study, we explored the role of CD11c(hi) dendritic cells (DCs) in regulating BBB integrity and JE progression using a conditional depletion model of CD11c(hi) DCs. Transient ablation of CD11c(hi) DCs resulted in markedly increased susceptibility to JE progression along with highly increased neuro-invasion of JEV. In addition, exacerbated JE progression in CD11c(hi) DC-ablated hosts was closely associated with increased expression of proinflammatory cytokines (IFN-β, IL-6, and TNF-α) and CC chemokines (CCL2, CCL3, CXCL2) in the brain. Moreover, our results revealed that the exacerbation of JE progression in CD11c(hi) DC-ablated hosts was correlated with enhanced BBB permeability and reduced expression of tight junction and adhesion molecules (claudin-5, ZO-1, occluding, JAMs). Ultimately, our data conclude that the ablation of CD11c(hi) DCs provided a subsidiary impact on BBB integrity and the expression of tight junction/adhesion molecules, thereby leading to exacerbated JE progression. These findings provide insight into the secondary role of CD11c(hi) DCs in JE progression through regulation of BBB integrity and the expression of tight junction/adhesion molecules.

Modulation of systemic and mucosal immunity against an inactivated vaccine of Newcastle disease virus by oral co-administration of live attenuated Salmonella enterica serovar Typhimurium expressing chicken interleukin-18 and interferon-α

Newcastle disease (ND) is a highly contagious disease of chickens causing significant economic losses worldwide. Due to limitations in the efficacy against currently circulating ND viruses, existing vaccination strategies require improvements, and incorporating immunomodulatory cytokines with existing vaccines might be a novel approach. Here, we investigated the systemic and mucosal immunomodulatory properties of oral co-administration of chicken interleukin-18 (chIL-18) and chicken interferon-α (chIFN-α) using attenuated Salmonella enterica serovar Typhimurium on an inactivated ND vaccine. Our results demonstrate that oral administration of S. enterica serovar Typhimurium expressing chIL-18 or chIFN-α provided enhanced systemic and mucosal immune responses, as determined by serum hemagglutination inhibition antibody and NDV Ag-specific IgG as well as NDV Ag-specific IgA in lung and duodenal lavages of chickens immunized with inactivated ND vaccine via the intramuscular or intranasal route. Notably, combined oral administration of S. enterica serovar Typhimurium expressing chIL-18 and chIFN-α significantly enhanced systemic and mucosal immunity in ND-vaccinated chickens, compared to single administration of S. enterica serovar Typhimurium expressing chIL-18 or chIFN-α. In addition, oral co-administration of S. enterica serovar Typhimurium expressing chIL-18 and chIFN-α provided enhanced NDV Ag-specific proliferation of peripheral blood mononuclear cells and Th1-biased cell-mediated immunity, compared to single administration of either construct. Therefore, our results provide valuable insight into the modulation of systemic and mucosal immunity by incorporation of immunomodulatory chIL-18 and chIFN-α using Salmonella vaccines into existing ND vaccines.

44: CD11chigh PDCA-1int/low dendritic cells regulates the differentiation of CD11b+Ly-6Chigh monocytes and permeability of blood–brain barrier to galvanize the immune privileged CNS to neuroinflammation

Although there were bunch of results showing the defined role of dendritic cells (DCs) in initiating adaptive host defense, their potential contributions to T-cell independent innate host defense and to subsequent immunopathology in immune privileged central nervous system (CNS) by neurotrophic virus is not completely defined. Notably, the potential roles of DC-monocyte interaction during neuroinflammatory course have not yet been addressed in depth. Here we found that CD11chighPDCA-1int/low DCs are essential to control the progression of neuroinflammation in immune privileged CNS upon infection of neurotrophic Japanese encephalitis virus (JEV), using selective ablation of CD11chighPDCA-1int/low DCs without altered CD11c[int]PDCA-1high plasmacytoid DC number, as manifested by increased CD11b+Ly-6Chigh monocyte infiltration, blood–brain barrier (BBB) permeability, viral burden, and cytokine expression. More surprisingly, CD11b+Ly-6Chigh monocytes were observed to have deleterious role in viral encephalitis, and selective ablation of CD11chighPDCA-1int/low DCs provided altered differentiation and function of infiltrated CD11b+Ly-6Chighmonocytes in CNS. Subsequently, CD11b+Ly-6Chigh monocytes generated in the absence of CD11chighPDCA-1int/low DCs elicited faster recruitment into inflamed CNS depending on CCR2, and augmented the severity of neuroinflammation caused by JEV infection, as were proved by several experimental models including adoptive transfer of CD11b+Ly-6Chigh monocytes using CCR2 KO and CCR2 KO · CD11c-DTR BM chimera. Our data identify for the first time an unrecognized role of CD11chighPDCA-1int/low DCs in maintaining the functional homeostasis of inflammatory CD11b+Ly-6Chigh monocytes and integrity of BBB, thereby affecting immunopathology in immune privileged CNS by neurotrophic virus.

TLR2/MyD88 pathway-dependent regulation of dendritic cells by dengue virus promotes antibody-dependent enhancement via Th2-biased immunity

Possible risk mediators in primary dengue virus (DenV) infection that favor secondary DenV infection to life-threatening dengue hemorrhagic fever (DHF) and shock syndrome (DSS) via antibody-dependent enhancement (ADE) have not yet been described. Here, DenV infection enhanced the expression of inflammatory mediators and activation molecules in dendritic cells (DCs) through TLR2/MyD88 pathway. TLR2 appeared to facilitate DenV infection in DCs that were less permissive than macrophages for viral replication. In experiments using separate evaluations of DenV-infected and uninfected bystander DCs, infected DCs showed impaired maturation accompanied with TLR2-dependent production of inflammatory cytokines, by which uninfected bystander DCs showed increased expression of co-stimulatory molecules. Differential phosphorylation of MAPK and STAT3 was also detected between DenV-infected and uninfected DCs. Furthermore, DenV infection stimulated Th2-polarized humoral and cellular immunity against foreign and DenV Ag via TLR2/MyD88 pathway, and DenV-infected DCs were revealed to facilitate Th2-biased immune responses in TLR2-dependent manner. TLR2/MyD88-mediated Th2-biased Ab responses to primary DenV infection increased the infectivity of secondary homotypic or heterotypic DenV via ADE. Collectively, these results indicate that TLR2/MyD88 pathway in DC-priming receptors can drive Th2-biased immune responses during primary DenV infection, which could favor secondary DenV infection to DHF/DSS via ADE.

Exacerbation of Japanese Encephalitis by CD11chi Dendritic Cell Ablation Is Associated with an Imbalance in Regulatory Foxp3+ and IL-17+CD4+ Th17 Cells and in Ly-6Chi and Ly-6Clo Monocytes

Japanese encephalitis (JE) is neuroinflammation characterized by uncontrolled infiltration of peripheral leukocytes into the central nervous system (CNS). We previously demonstrated exacerbation of JE following CD11chi dendritic cell (DC) ablation in CD11c-DTR transgenic mice. Moreover, CD11chi DC ablation led to abnormal differentiation of CD11b+Ly-6Chi monocytes and enhanced permeability of the blood-brain barrier (BBB), resulting in promoting the progression of JE. Here, we examined changes in lymphoid and myeloid-derived leukocyte subpopulations associated with pro- and anti-inflammation during JE progression. The analyses of this study focused on regulatory CD4+Foxp3+ regulatory T cells (Tregs), IL-17+CD4+ Th17 cells, and CD11b+Ly-6Chi and Ly-6Clo monocytes. CD11chi DC ablation resulted in the accumulation of IL-17+CD4+ Th17 cells in the CNS, thereby leading to lower ratio of Tregs to Th17 cells. This result was corroborated by the higher expression levels of IL-17 and RORγT in CD4+ T cells from the brains of CD11chi DC-ablated mice. In addition, CD11chi DC-ablated mice showed higher frequency and total number of inflammatory CD11b+Ly-6Chi monocytes, whereas CD11b+Ly-6Clo monocytes were detected with lower frequency and total number in CD11chi DC-ablated mice. Furthermore, CD11chi DC ablation altered the phenotype and function of CD11b+Ly-6Clo monocytes, resulting in lower levels of activation marker and anti-inflammatory cytokine (IL-10 and TGF-β) expression. Collectively, these results indicate that CD11chi DC ablation caused an imbalance in CD4+ Th17/Treg cells and CD11b+Ly-6Chi/Ly-6Clo monocytes in the lymphoid tissue and CNS during JE progression. This imbalanced orchestration of pro- and anti-inflammatory leukocytes following CD11chi DC ablation may contribute to the exacerbation of JE.